The overall goal of this research proposal is to find genetic variation that explains differences in male fertility. There is a high incidence of unexplained male infertility in humans, which can be a clinical sign that precedes many more devastating diseases, including prostate cancer and testicular cancer. In this era of assisted reproductive technologies, mutations that lead to male infertility are being passed to the next generation at an ever increasing pace, making it critically important to understand the genes that affect male reproduction. To link genetic with phenotypic divergence, we take a novel approach and combine evolutionary population genetic analyses with laboratory investigations of phenotypic variation in male reproductive traits. We focus on wild mice species that differ in their mating ecology to dissect the genetics of male reproduction. With an evolutionary perspective, we can detect mutations with much subtler effects on phenotype than can be detected solely in the laboratory. We have four specific aims: (1) Genome Evolution - to sequence 400 seminal fluid genes and 400 spermatogenesis genes from wild caught individuals sampled from eight species of Mus, including population level samples from M. domesticus and M. spretus, two species with very different mating ecologies and presumably selective regimes. Analyzing these data in an evolutionary population genetics framework will lead us to genes that have undergone adaptive evolution. (2) Transcriptome Evolution - to perform sequence-based quantification of gene expression across three distinct developmental timepoints of spermatogenesis. These data will extend our understanding of the evolution of gene regulation during spermatogenesis and allow us to identify patterns of expression indicative of adaptive evolution. (3) Proteome Evolution - to quantify the relative abundance of the major seminal fluid proteins, to test a novel hypothesis that adaptation occurs via shifts in protein abundance in addition to changes in primary sequence or gene expression. (4) Phenome Evolution - to intensively phenotype a newly established set of wild derived inbred strains to investigate the divergence of male reproductive traits across three species with distinct mating ecologies. These four specific aims synergize multiple levels of biological information to gain fundamental insights into the genetic basis of male reproduction. PUBLIC HEALTH RELEVANCE: We propose to find the genetic basis of male reproductive variance, using a novel combination of evolutionary and laboratory studies. Our specific aims are to study adaptive evolution at the genomic, transcriptomic, and proteomic levels, and then to make the critical next step of linking molecular variation at these three levels to variation at a large set of male reproductive traits. We focus on wild mice species that differ in mating ecology, providing a powerful complement to classical inbred strains and other existing resources.